The three isoforms of TGF-beta expressed in mammals are functionally interchangeable in most in vitro assay systems, but they have distinctive activities on certain cells and in direct binding to receptors. To define specific regions of the TGF-beta molecule responsible for these differences in activities, we developed a system for expression and purification of recombinant TGF-betas engineered to have either mutations or substitutions of homologous regions of different isoforms, based on the three-dimensional structure of TGF-beta. Using recombinant soluble TGF-beta type II receptor, we have identified a region of TGF-beta1 (amino acids 92-96) involved in isoform-specific receptor binding. In related research, we are identifying down-stream mediators of the signal transduction pathways of the TGF-beta serine-threonine kinase receptors. Using clues from fly genetics, we have now identified a family of eight distinct proteins (Smad 1-8) related to Drosophila Mad, a cytoplasmic mediator of signals from a TGF-beta family ligand, dpp. Thus far we have demonstrated that Smad1 or a related protein rapidly becomes phosphorylated on serine and threonine when cells are treated with TGF-beta, and that Smad4, identical to a candidate tumor suppressor for pancreatic cancer DPC4, can restore TGF-beta signal transduction pathways in tumor cells null for Smad4, supporting our previous hypothesis that intermediates in TGF-beta signal transduction pathways will have tumor suppressor activity. By constructing chimeric Smad proteins, we have further shown that the ligand-dependency of the Smad proteins resides in their unique proline-rich linker regions. Ongoing studies are focused on identification of specific phosphorylation sites in Smads 1 and 4 to enable construction of Smad proteins mutated in these positions to evaluate the role of phosphorylation in nuclear translocation and signal transduction. We also have a putative 100 kD protein which binds specifically to the middle region of Smad1 and are attempting to sequence this protein to enable cloning, expression, and determination of its biological activity. We are also attempting to define possible synergistic interactions of distinct Smad proteins in mediation of signals from TGF-beta receptors.